MEDICINA - Volumen 79 - N° 6/1 - NÚMERO ESPECIAL 80 ANIVERSARIO, 2019546

SPECIAL ARTICLE - REVIEW

ISSN 1669-9106

BIOMARKERS FOR ALZHEIMER´S DISEASE. WHERE WE STAND AND WHERE WE ARE HEADED

PATRICIO CHREM MÉNDEZ1, EZEQUIEL SURACE2, YANINA BÉRGAMO3, ISMAEL CALANDRI1,SILVIA VÁZQUEZ3, GUSTAVO SEVLEVER2, 4, RICARDO F. ALLEGRI1, 5

1Centro de Memoria y Envejecimiento, 2Laboratorio de Biología Molecular, 3Centro de Imágenes Moleculares (CIM),4Banco de Cerebro, Instituto de Investigaciones Neurológicas, FLENI, Buenos Aires, Argentina,

5Departamento de Neurociencias, Universidad de la Costa (CUC), Colombia

Abstract Alzheimer disease (AD) is one of the major unresolved health burdens accompanying the increase in lifeexpectancy.Thegreatparadigmshift for thisdiseasehasresultedfromfindingamyloidde-position and neurobrillary degeneration 20 years and 10 years, respectively, prior to onset of the typical clinical memory loss symptoms. The advent of AD biomarkers has enabled amolecular definition of AD,making theclinicaldefinitionalmostdispensable.VarioustypesofADbiomarkersareavailableinourcountry.Eachbiomarkerreflectsaparticularprocessandstageof thedisease.Althoughcostsrestrict theiruse, thebiomarkeranalysismaybejustifiedincertainclinicalscenarios,suchasanearlyonsetoranatypicalpresentationofthedisease.Today, the usefulness of biomarkers in AD clinical research is beyond question. Furthermore, the introduction of biomarkersintomedicalpracticehasledtosignificantchangesintherapeuticinterventions,evenintheabsenceof disease-modifying drugs.

Key words: Alzheimer disease, biomarkers, preclinical, neuroimaging, molecular diagnostic techniques

Resumen Biomarcadores de enfermedad de Alzheimer. Dónde estamos y hacia dónde vamos. La en- fermedaddeAlzheimer(EA)esunode losmayoresflagelosaúnnoresueltosqueacompañanalaumentodelaexpectativadevida.Elgrancambiodeparadigmaenlosúltimosañosfueconsecuenciadedes-cubrirqueeldepósitoamiloideosepresentahasta20añosantes,yladegeneraciónneurofibrilarhasta10añosantes, de que aparezca la sintomatología clínica típica de pérdida de memoria. La aparición de los biomarcadores permitió reestructurar el concepto de laEA, intentándose llegar a una definiciónmolecular de lamisma casiprescindiendo de la emblemática clínica. Existen distintos tipos de biomarcadores de EA disponibles en nuestro país.Cadaunonoshabladeunprocesoyunmomentodistintode laenfermedad.Aunquesuusoclínicoaúnseencuentrarestringidoporcuestionesdecostos,existenescenariosparticularesendondesísejustifica,casisiempre en relación a presentaciones clínicas atípicas o de comienzo muy temprano. Sin embargo, hoy en día ya nadie discute que son imprescindibles en investigaciones clínicas sobre EA. La incorporación de biomarcadores enlaprácticamédicahageneradocambiossignificativosenlaintervenciónterapéuticadelospacientes,inclusoenuncontextoenelquetodavíanohaymedicamentosmodificadoresdelaenfermedad.

Palabras clave: biomarcadores de la enfermedad de Alzheimer, etapas preclínicas, neuroimágenes, diagnóstico molecular

Received:27-V-2019 Accepted:10-VI-2019

Postal address: PatricioChremMendez,FLENI,Montañeses2325,1428 Buenos Aires, Argentina e-mail: pchremmendez@fleni.org.ar

The Alzheimer disease (AD) is one of the main un-resolved health burdens accompanying the increase in life expectancy and has become a crucial public health problem1. One out of every two individuals over the age of 80 will develop the disease with the ensuing impact on their family and society2.

The main pathophysiological mechanism underlying AD involves increase in Aβ peptide species, ultimately leadingtoextracellularamyloiddepositsandneurofibril-lary degeneration, secondary to intraneuronal abnormal tau protein hyperphosphorylation. In 2012, Bateman et al.3

showedthatamyloiddepositsandneurofibrillarydegen-eration were present 20 and 10 years before the onset of memory decline, respectively.

Asaresultofthesefindings,thecourseofADwasdivided into three stages –presymtomatic, mild cogni-tiveimpairment,anddementia−withthepresymtomaticphase currently attracting major research efforts on AD pathophysiology, treatment, and prevention.

Prior to the introduction of biomarkers, the clinical di-agnosis (NINCDS-ADRDA criteria) was limited to possible orprobableAD,giventhatdiagnosiswasonlyconfirmedin post-mortem brain tissue histopathology 4.

Biomarkersaredefinedasobjective,quantifiablepa-rameters which allow in vivo assessment of pathophysi-ological disease traits. Current biomarkers for AD include: 1) Aβ1-42 , total tau and phosphorylated tau assay in cere-brospinalfluid(CSF);2)structuralneuroimagingstudies

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suchasbrainMRIandhippocampalvolumeanalysis;3)functional neuroimaging of metabolic activity such as 18F-fluorodesoxyglucose(FDG)PETandprotein-identifyingneuroimaging such as amyloid PET and tau PET.

Basedon theapplicationof thesespecificbiomark-ers, the US National Institute of Aging and Alzheimer’s Association revised the accepted diagnostic criteria for AD5-8, which led to two important changes. First, the use of biomarkers allowed a formal separation of the different disease stages to include mild cognitive impairment, thus providinggreatersensitivityandspecificitytothedetectionof early AD. Second, the application of biomarker results allowed to achieve higher diagnostic certainty, in relation to the underlying neuropathological changes present in AD.

In2018,anewbiomarker-basedbiologicalclassifica-tion,theA/T/N(Amyloid/Tau/Neurodegeneration)system,was published9, in which “A” refers to the presence of β-amyloid biomarker (detected on amyloid PET or as-saying CSF Aβ42 level); “T” refers to thevalueofa taubiomarker (measured in CSF phospho-tau assay, or on tau PET);and“N”referstobiomarkersofneurodegenerationor neuronal injury (evaluated on [18F]-fluorodeoxyglucose-PET, structural MRI, or measuring total tau in CSF). This classificationallowsapathophysiologicalcategorizationand a clearer prediction of patient outcome9.

The introduction of biomarker results has profoundly influencedADdiagnosis,prognosisandtreatment,sinceit allows the detection of very early stages in individuals presenting mild AD symptoms without dementia (prodro-mal AD), or even at pre-symptomatic stages.

Thus, with pharmacological treatments becoming avail-able for very early stages of the AD, the major challenge becomesfindingsimplerbiomarkers.Wehopethatinthenot too distant future the value of their potential applica-tion extends to all clinical scenarios for which they may prove useful.

Alzheimer´s disease biomarkers in fluids

A better understanding of the disease mechanisms in-volved in AD has allowed the development of different types of fluid biomarkers.CSFhas becomea primaryviable source, given its close contact with the CNS at the extracellular compartment level. However, because of the invasive nature of the spinal tap, patients are reluctant to undergo testing, and repeated sampling is poorly toler-ated, which is why biomarkers in blood are now being investigated and validated10.

Three key CSF biomarkers have been included in several guidelines and research manuals. These are: β-amyloid 42 (Aβ1-42), total tau (t-tau) and threonine18-phosphorylated tau (p-tau). Altered CSF levels of any of these are recognized diagnostic evidence of underlying brain disease compatible with AD8. Added prognostic

value has also been reported for these molecules as evidence of disease progression in individuals who remain cognitively intact8 as well as in individuals with mild cognitive impairment6. Also, Aβ1-42 levels may help to distinguish AD from other clinical conditions such as frontotemporal dementia.

In recent years, new biomarkers have been described as related to other pathophysiological aspects such as vascular dysfunction, neuronal and synaptic integrity andneuroinflammation, to namea few. In this regard,neurofilament-lightchain(NfL),anintermediatefilamentof the neuronal cytoskeleton, which is abundant in axons, has been recognized as a marker of neuronal damage, increasing in both CSF and blood as a result of different neurodegenerative diseases11-14. Although not specificfor AD, NfL blood levels could be useful for screening purposes. When high NfL levels are detected, subjects could then be tested for known AD biomarkers, namely Aβ1-42, t-tau or p-tau in CSF, or PET PiB.

Other biomarkers, such as neurogranine (a marker of synapsedysfunction)ormarkersof inflammation likeT-REM2 and YKL-40, require more exhaustive validation before they are included in the panel of accepted AD biomarkers12.

Aside from their role in diagnosis, biomarkers could soon become indispensable tools for the development of future AD therapies. Currently, their use in clinical trials improves the classificationof participants according tothe underlying disease, allows staging the disease more precisely and also allows a better and earlier evaluation of treatment response13.

Prior to implementation, new biomarkers will require validation for different conditions or stages and different applications such as screening, diagnosis, treatment monitoring, among others15.

Alzheimer´s disease biomarkers in neuroimaging

Morphological imaging, using both magnetic resonance (MRI) and computed tomography (CT), as well as mo-lecular methods like positron emission tomography (PET) and single photon emission tomography (SPECT), play an important role in early diagnosis and in non-invasive in vivo follow up of patients with neurodegenerative syndromes. Brain deposits of the abnormal proteins Aβ and tau can be detected using PET and neuronal dysfunction measured byanalyzingbrainglucosemetabolism(FDG).Cerebralperfusion evaluated by SPECT shows good correlation with metabolic changes.

AD is the most prevalent neurodegenerative disease and presents a particular molecular and structural neuro-imagingprofile,inwhichcorticalandextraneuronalamy-loid deposits precede the emergence of clinical symptoms

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by 20 years. PET using amyloid-labeling tracers (11C-PIB, 18F-Flutemetamol and 18FAV45,amongothers),representsa sensitive tool for early detection of abnormal brain de-posits in the areas most often affected, namely the bilateral frontal and parietal lobes, the lateral temporal cortex and striatal regions. Although detection of amyloid implies greater disease risk, or increased diagnostic certainty, it is important to note that amyloid deposits are present in up to 30% of cognitively normal individuals.

The tauprotein is found inneurofibrillary tanglesatintraneuronal level, and its hyperphosphorylated forms arespecifictoAD.OnPETimaging,differenttracersstillunder development can be detected with varying degrees of sensitivity and specificity. Flortaucipir (18FAV1451)has been tested in our country in the ADNI-Arg cohort of patients with clinical diagnosis of AD followed for 5 years (Alzheimer Disease Neuroimaging Initiative – Ar-gentina); thestudyshowedhigherconcentration in themesial temporal lobe. Neuronal dysfunction explored us-ing 18FDG-PET,amarkerofneurodegeneration,showedbilateral areas of hypometabolism in the temporal lobe, the precuneus, the posterior cingulated cortex, and the parietallobe.Altogether,thesefindingstendtoshowthefollowing chronological sequence of tracer uptake during preclinicalphases:firstamyloiddeposition,thentaude-position, and later neuronal dysfunction8. As an example, Fig. 1 shows images of brain PET scans illustrating the biomarkerfindingscharacteristicoftheATNclassification.

Structural brain MRI reveals the presence of reduced hippocampal volume, aswell as area-specific corticalatrophy (parahippocampal gyrus, amygdala, superior, medial and inferior temporal gyri, superior parietal lobe and posterior cingulate cortex). Both are considered biomark-ers of neurodegeneration generally appearing later than others, that is, during symptomatic stages of the disease. Aspecificsoftwareisusedtomeasurefindingscomparedto quantitative values of standardized digital atlas images, which are available in most local imaging centers.

The functional imaging technique most recently incor-porated is the resting state MRI (rs-fMR), in which BOLD signal detection generates resting state images (without any type of activity). This allows “connections” between dif-ferent areas to be studied by correlating temporal neuronal activity between different areas of the brain cortex. The areas of the greatest synchronicity or correlation represent closely linked neuronal networks. To date, rs-fMRI results have shown different connectivity patterns in AD patients compared to normal controls, as well as in patients with mild cognitive impairment and even at preclinical stages of the disease16.

In summary, a series of sequential and concurrent events need to be taken into consideration, both spatially and chronologically, which include detection of Aβ42 and phospho-Tau, volumetric analysis, hypometabolic areas and connectivity disruption. Imaging techniques provide

information on different “stages or phases” of the AD continuum, generating new opportunities for eventual treatment interventions.

Clinical utility of biomarkers inAlzheimer´s disease

Based on all of the above, it seems clear that the biomark-ers have improved the diagnosis of AD beyond clinical findings,andhaveshownthatthepreclinicalstagesofthedisease may in fact last much longer than the symptomatic ones3,8,9,13,15.Asanexample,Fig.2showsA/T/Nresultsin theArgentineADNIcohort (normalcontrols,n=14;earlymildcognitiveimpairmentpatients,n=10;latemildcognitive impairment patients n =1 3, and patients with dementia of the Alzheimer type, n = 1217).

Today, the value of this classification in clinical tri-als and clinical research is beyond question. However, numerous limitations hinder its application in routine clinical practice, in particular for developing countries in Latin-American,mainlyduetofinancialconstraints,limitedexperience and validation, and restricted clinical access for most of the region. A recent survey has shown however, that it is nevertheless already being used in Argentina, Brazil, Colombia, Chile, Mexico and Uruguay, among other countries18.

If no disease-modifying drug is available, what is the point of applying biomarkers in clinical practice? Several papers have recommended their use in specific situa-tions19. There is general agreement that in early-onset disease (under 65 years of age) as well as in atypical forms of AD, which often also present before 65, biomark-ers could be useful. Less anosognosia is present in this age group, which is more concerned on diagnosis and still atveryproductivestagesoflife.Diagnosticconfirmationwould allow making life-changing decisions and improve differential diagnosis with other radically different types of disease. Finally, genetic implications may exist for other family members, (i.e. autosomal dominant forms of AD). One could also add groups of patients in whom AD is suspected, but who present pre-morbid psychiatric condi-tions,whichincreasesignificantlythediagnosticdifficulty.

Althoughclinicalsyndromesexistwithsignificantover-lappingregardingamyloidfindings(highpretestprobabil-ity, i.e. for posterior cortical atrophy), the opposite occurs in other clinical conditions (pretest likelihood is low), hence the value of biomarkers19.

ThefindingsoftheIDEASprotocol,conductedon18000 imaging studies in US centers to evaluate the clinical utility of amyloid PET results, have recently been pub-lished20.Briefly,thestudyshowedthattheparticipatingphysiciansmodifiedprescriptionsorchangedtheirclinicalmanagement in 60% of the patients based on imaging results. This shows that vis-à-vis the diagnostic uncertainty

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• A-T-(N-)PETimagesofamyloidandtaudonotshowtraceruptake.NormalmetabolismonFDGPET.Imagescorrespondto normal subjects

• A+T-(N-) PET image is positive for amyloid, cortical tracer uptake is visible. Tau PET is negative and metabolism normal onFDG.Theseimagescorrespondtotheinitialstageofcerebralamyloidosisinpre-symptomaticAD

• A+T+(N-)PETimageispositiveforamyloidandtau,corticaluptakeisseenforboth.PETFDGshowsnormalmetabolism.These images correspond to the second stage of pre-symptomatic AD with amyloid and tau deposits

• A+T+(N+)ThiscaseshowscorticalamyloidandtaudepositsandposteriorhypometabolismonFDGPET.Typicalimagesin AD patients

• A+T-(N+) This panel shows anterior cortical positivity on amyloid PET, Tau PET is negative y and there is posterior hypo-metabolismmainlyontherightsideonFDGPET.ThiscaseiscompatiblewithADassociatedtonon-ADpathology

• A-T+(N-) No amyloid deposits are observed. PET tau shows posterior cortical tau deposits predominantly on the left side andnormalFDG-PET.ThisisobservedindegenerativediseaseotherthanAD

• A-T-(N+)AmyloidPETandtauarenegative.FDGPETshowsanteriorhypometabolismcompatiblewithnon-ADdegene-rative pathology

• A-T+(N+)AmyloidPETisnegativebuttauPETispositiveinthefrontalandposteriorcortex.FDG-PETshowsleftanteriorand posterior hypometabolism compatible with non-AD pathology

Fig. 1.–ATNclassificationbasedonbrainPET (amyloid, tauandFDG),FLENINeuroimaging database

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inAD,biomarkerresultsexertsignificantinfluenceondailymedical practice.

Discussion

ADhadpreviouslybeendefinedbasedonAloisAlzheim-er’soriginaldescriptionofneuropathologicalfindingsinautopsy material. The diagnosis was based on the detailed presence, density and distribution of the characteristic lesions, namely extracellular amyloid plaque and intraneu-ronalneurofibrillarydegeneration.Thesecriteriawerelaterrefinedandquantifiedinanattempttoincludeotherlesionsthat usually coexist in the aging brain and affect cognition.

The discovery of biomarkers which can report in vivo on the presence of these deposits has radically changed neurodegeneration diagnosis. However, as is common in biomedicine, technical advances come together with prob-ably even more relevant conceptual changes. In the case ofAD,thishasledtoanewdefinitionofthedisease,sepa-ratingitfromdementiaasasingleclinicalfinding,whichcan develop during the course of several other concurrent diseases. Diagnosis today is determined by detection of amyloid, T-tau and P-tau. It is not known whether these abnormal proteins actually cause the disease, but they areneverthelesstheirdefiningfeature.Proteindeposits

makeADauniqueandspecificneurodegenerativedisor-der, separating it from other conditions causing dementia. This differentiation is key, since it allows examination of chronological events leading to clinically evident effects on cognition as a continuum, which includes intact subjects amenable to potential treatment strategies. In the not too distant future, taxonomic consequences resulting from biomarker use are foreseeable, linking the nosology to an underlying molecular abnormality, which in turn, may become a potential objective for targeted therapies.

Conflict of interest: None to declare

References

1. World Health Organization and Alzheimer’s Disease In-ternational. Dementia: a public health priority. In: https://www.who.int/mental_health/publications/dementia_re-port_2012/en/;accessedMarch2019.

2. AllegriRF,VázquezS,SevleverG.EnfermedaddeAl-zheimer: Nuevos paradigmas, 2da. ed. Buenos Aires: Editorial Polemos, 2018.

3. Bateman RJ, Xiong C, Benzinger TL, et al. Clinical and biomarker changes in dominantly inherited Alzheimer’s disease. N Engl J Med2012;367:795-804.

4. McKhannG,DrachmanD,FolsteinM,KatzmanR,PriceD, Stadlan EM. Clinical diagnosis of Alzheimer’s disease: report of theNINCDS-ADRDAWorkGroup under the

ADNI: Alzheimer Disease Neuroimaging Initiative; NC = normal control; e-MDI = early mild cognitive impairment; l-MCI = late mild cognitive impairment; DAT = dementia Alzheimer type; A /T /N Amyloid, Tau; Neurodegeneration

% of patients with each ATN subtype in the Argentine ADNI- cohort (Alzheimer Disease Neuroimaging Initiative - Argentina)

Fig.2.–ATNClassificationoftheArgentineADNIcohortatFLENI

BIOMARKERS IN ALZHEIMER´S DISEASE 551

auspices of Department of Health and Human Services Task Force on Alzheimer’s Disease. Neurology1984;34:939-44.

5. Jack CR Jr, Albert MS, Knopman DS, et al. Introduction to the recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement 2011;7:257-62.

6. Albert MS, DeKosky ST, Dickson D, et al. The diagnosis of mild cognitive impairment due to Alzheimer’s disease: recommendations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guide-lines for Alzheimer’s disease. Alzheimers Dement 2011;7:270-9.

7. McKhannGM,KnopmanDS,ChertkowH,etal.Thediag-nosis of dementia due to Alzheimer’s disease: recommen-dations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement2011;7:263-9.

8. SperlingRA,AisenPS,BeckettLA,etal.Towarddefiningthe preclinical stages of Alzheimer’s disease: recommen-dations from the National Institute on Aging-Alzheimer’s Association workgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement2011;7:280-92.

9. JackCRJr,BennettDA,BlennowK,etal.NIA-AA Re-searchFramework:TowardabiologicaldefinitionofAl-zheimer’s disease. Alzheimers Dement2018;14:535-62.

10. Hansson O, Zetterberg H, Buchhave P, Londos E, Blennow K, Minthon L. Association between CSF biomarkers and incipient Alzheimer’s disease in patients with mild cogni-tive impairment: a follow-up study. Lancet Neurol2006;5: 228-34.

11. Niikado M, Chrem-Méndez P, Itzcovich T, et al. Evalua-tionofcerebrospinalfluidneurofilamentlightchainasaroutine biomarker in a memory clinic. J Gerontol A Biol Sci Med Sci2019;74:442-5.

12. Hampel H, O’Bryant SE, Molinuevo JL, et al. Blood-based biomarkers for Alzheimer disease: mapping the road to the clinic. Nat Rev Neurol2018;14:639-52.

13. HampelH,VergalloA,PerryG,ListaS.TheAlzheimerPrecision Medicine Initiative. Alzheimer Precision Medicine Initiative (APMI). J Alzheimers Dis 2019;68: 1-24.

14. PreischeO,SchultzSA,ApelA,etal.Serumneurofila-ment dynamics predicts neurodegeneration and clinical progression in presymptomatic Alzheimer’s disease. Nat Med2019;25:277-83.

15. VeitchaDP,WeinerMW,AisengPS,etal.Understanding disease progression and improving Alzheimer’s disease clinical trials: Recent highlights from the Alzheimer’s Dis-ease Neuroimaging Initiative. Alzheimers Dement2019;15: 106-52.

16. Buckner RL, Andrews-Hanna JR, Schacter DL. The brain´s default network: anatomy, function, and relevance to disease. Ann NY Acad Sci2008;1124:1-38.

17. AllegriRF,PertierraL,CohenG,etal.Abiologicalclas-sification for Alzheimer’s disease - Amyloid, Tau andNeurodegeneration (A/T/N): results from theArgentine-Alzheimer’s Disease Neuroimaging Initiative. Int Psycho-geriatr2019;12:1-2.

18. Parra MA, Baez S, Allegri R, et al. Dementia in Latin America: Assessing the present and envisioning the future. Neurology2018;90:222-31.

19. ChremMéndezP,CohenG,RussoMJ,etal.Concordance between 11C-PIB-PET and clinical diagnosis in a memory clinic. Am J Alzheimers Dis Other Demen2015;30:599-606.

20. RabinoviciGD,GatsonisC,ApgarC,etal.Association of amyloid positron emission tomography with subsequent change in clinical management among medicarebenefi-ciaries with mild cognitive impairment or dementia. JAMA 2019;321:1286-94.